Micro-HPLC of proteins using a novel monolithic stationary phase with a gradient of functionalities prepared via photoinitiated grafting

The use of photografting for the functionalization of porous poly(butyl methacrylate-co-ethylene dimethacrylate) monolithic columns enabled the preparation of columns with a homogeneously grafted poly(2-acrylamido-2-methyl-1-propanesulfonic acid, AMPS) chains. Recently, novel monolithic columns with a longitudinal gradient of concentration of functional groups were prepared and characterized using electron probe microanalysis and the separation performance evaluated in CEC. We demonstrated that these monolithic columns afforded higher separation efficiency and peak capacity compared to their homogeneously grafted counterparts. Consequently, the application of monolithic columns with this new type of chemistry enabled rapid and efficient separations. In this study, we now demonstrate the applicability of the new type of monolithic columns in µ-HPLC. For example, the separation of proteins in ion-exchange mode was studied. Optimization of the chromatographic conditions such as the shape of the mobile phase gradient and the flow rate allowed a rapid separation of four proteins in a short period of time. We also found that the stationary phase involving the gradient of functionalities exhibited high selectivity. These separation methods can be useful in the proteomic research.